Severe asthma in horses is associated with increased airway innervation.

BACKGROUND: Altered innervation structure and function contribute to airway hyperresponsiveness in human asthma, yet the role of innervation in airflow limitation in asthma in horses remains unknown. HYPOTHESIS: To characterize peribronchial innervation in horses with asthma. We hypothesized that airway innervation increases in horses with asthma compared with controls. ANIMALS: Formalin-fixed lung samples from 8 horses with severe asthma and 8 healthy horses from the Equine Respiratory Tissue Biobank. Ante-mortem lung function was recorded. METHODS: Blinded case-control study. Immunohistochemistry was performed using rabbit anti-s100 antibody as a neuronal marker for myelinating and non-myelinating Schwann cells. The number and cumulative area of nerves in the peribronchial region and associated with airway smooth muscle were recorded using histomorphometry and corrected for airway size. RESULTS: Both the number (median [IQR]: 1.87 × 10-5 nerves/µm2 [1.28 × 10-5 ]) and the cumulative nerve area (CNA; 1.03 × 10-3 CNA/µm2 [1.57 × 10-3 ]) were higher in the peribronchial region of horses with asthma compared with controls (5.17 × 10-6 nerves/µm2 [3.76 × 10-6 ], 4.14 × 10-4 CNA/µm2 [2.54 × 10-4 ], Mann-Whitney, P = .01). The number of nerves within or lining airway smooth muscle was significantly higher in horses with asthma (4.47 × 10-6 nerves/µm2 [5.75 × 10-6 ]) compared with controls (2.26 × 10-6 nerves/µm2 [1.16 × 10-6 ], Mann-Whitney, P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Asthma in horses is associated with greater airway innervation, possibly contributing to airway smooth muscle remodeling and exacerbating severity of the disease.

Engineered Curli Nanofilaments as a Self-Adjuvanted Antigen Delivery Platform.

Proteinaceous nanoparticles constitute efficient antigen delivery systems in vaccine formulations due to their size and repetitive nature that mimic most invading pathogens and promote immune activation. Nonetheless, the coadministration of an adjuvant with subunit nanovaccines is usually required to induce a robust, long-lasting, and protective immune response. Herein, the protein Curli-specific gene A (CsgA), which is known to self-assemble into nanofilaments contributing to bacterial biofilm, is exploited to engineer an intrinsically immunostimulatory antigen delivery platform. Three repeats of the M2e antigenic sequence from the influenza A virus matrix 2 protein are merged to the N-terminal domain of engineered CsgA proteins. These chimeric 3M2e-CsgA spontaneously self-assemble into antigen-displaying cross-ß-sheet nanofilaments that activate the heterodimeric toll-like receptors 2 and 1. The resulting nanofilaments are avidly internalized by antigen-presenting cells and stimulate the maturation of dendritic cells. Without the need of any additional adjuvants, both assemblies show robust humoral and cellular immune responses, which translate into complete protection against a lethal experimental infection with the H1N1 influenza virus. Notably, these CsgA-based nanovaccines induce neither overt systemic inflammation, nor reactogenicity, upon mice inoculation. These results highlight the potential of engineered CsgA nanostructures as self-adjuvanted, safe, and versatile antigen delivery systems to fight infectious diseases.

Differences in Parents and Teachers' Perceptions of Behavior Manifested by Gifted Children with ADHD Compared to Gifted Children without ADHD and Non-Gifted Children with ADHD Using the Conners 3 Scale.

The potential for the misdiagnosis of giftedness as attention deficit/hyperactivity disorder (ADHD) has been well documented, as has the clinical diagnostic profile of individuals with both giftedness and ADHD. This study aimed to examine parents' and teachers' responses to the Conners 3 behavioral rating scale of gifted students with ADHD compared to gifted students without ADHD and non-gifted students with ADHD. Ninety-two children aged 6 to 16 years were included in the study. On the basis of clinical assessments utilizing the K-SADS, the WISC-V, and other neurocognitive tests, the students were split into three groups: gifted/ADHD (n = 35), ADHD (n = 35), and gifted (n = 22). The results revealed that mothers', fathers', and teachers' responses to the Conners 3 rating scale distinguished well between the gifted group and the other two groups, but not between the gifted/ADHD and ADHD groups. The learning difficulties observed by teachers was the most significant element that distinguished gifted/ADHD students from non-gifted ADHD students. Other results indicated that mothers and fathers reported more inattention problems in their gifted/ADHD children than teachers. Additionally, mothers tended to observe more learning and executive function problems in their gifted/ADHD children than teachers did. These findings highlight the importance of multiple informants complementing each other in the assessment process for ADHD in a gifted context to counteract the masking effect between giftedness and ADHD.

Self-Assembly of Flagellin into Immunostimulatory Ring-like Nanostructures as an Antigen Delivery System.

Proteinaceous nanoparticles represent attractive antigen carriers for vaccination as their size and repetitive antigen displays that mimic most viral particles enable efficient immune processing. However, these nanocarriers are often unable to stimulate efficiently the innate immune system, requiring coadministration with adjuvants to promote long-lasting protective immunity. The protein flagellin, which constitutes the primary constituent of the bacterial flagellum, has been widely evaluated as an antigen carrier due to its intrinsic adjuvant properties involving activation of the innate immune receptor Toll-like receptor 5 (TLR5). Although flagellin is known for its ability to self-assemble into micron-scale length nanotubes, few studies have evaluated the potential usage of flagellin-based nanostructures as immunostimulatory antigen carriers. In this study, we reported for the first time a strategy to guide the self-assembly of a flagellin protein from Bacillus subtilis, Hag, into lower aspect ratio nanoparticles by hindering non-covalent interactions responsible for its elongation into nanotubes. We observed that addition of an antigenic sequence derived from the influenza A virus (3M2e) at the C-terminus of this flagellin, as opposed to positioning the epitope into mid-sequence, precluded filament elongation and resulted in low aspect ratio ring-like nanostructures upon salting-out-induced self-assembly. These nanostructures displayed the antigen at their surface and shared morphological and structural characteristics with flagellin nanotubes, with a diameter of approximately 12 nm, and an α-helix-rich secondary structure. Flagellin ring-like nanostructures were efficiently internalized by antigen-presenting cells, and avidly activated the TLR5 in vitro as well as the innate and adaptive immune responses. Intranasal immunization of mice with these nanostructures resulted in the potentiation of the antigen-specific antibody response and protection against a lethal infection with the influenza A virus, illustrating the potential of these intrinsically immunostimulatory nanostructures as antigen carriers.

Recombinant Bacillus subtilis flagellin Hag is a potent immunostimulant with reduced proinflammatory properties compared to Salmonella enterica serovar Typhimurium FljB.

Flagellin constitutes a potential adjuvant for vaccines owing to its robust immunostimulatory properties. However, clinical trials have revealed that flagellin derived from Salmonella enterica serovar Typhimurium induces high levels of proinflammatory markers and substantial adverse effects. The flagellin from Bacillus subtilis, Hag, shares high sequence homology with Salmonella FljB within the D0 and D1 domains responsible for TLR5 engagement, while the D2 and D3 domains associated with an off-target immune response are absent. Accordingly, we compared the immunostimulatory and proinflammatory properties of Hag with FljB by harnessing an epitope from the matrix 2 protein (M2e) of the influenza virus. Both flagellins engaged TLR5, with FljB showing a 2.5-fold higher potency than Hag. Mice inoculation showed a robust FljB- or Hag-induced M2e-specific antibody response, with Hag demonstrating a decreased secretion of proinflammatory markers and reduced weight loss. This study revealed that flagellin Hag is a potent immunoadjuvant with reduced proinflammatory properties.

Assessment of muscular strength and functional capacity in the juvenile and adult myotonic dystrophy type 1 population: a 3-year follow-up study.

INTRODUCTION: Myotonic dystrophy type 1 (DM1) is a progressive, multisystemic, and autosomal dominant disease. Muscle wasting and weakness have been associated with impaired functional capacity and restricted social participation in affected individuals. The disease's presentation is very heterogenous and its progression is still under-documented. OBJECTIVE: The aim of the study was to document the progression of muscular strength and functional capacity in the DM1 population over a 3-year period. METHODS: Twenty-three individuals with juvenile or adult phenotypes of DM1 were recruited to complete clinical assessments in 2016 and 2019. Maximal isometric muscle strength (MIMS) was evaluated with quantified muscle testing and functional capacity was evaluated with the Mini-BESTest, the 10-m walk test at comfortable and maximal speeds, the Timed Up and Go and the 6-min walk test. Participants also completed three questionnaires: DM1-Activ, Upper Extremity Functional Index and Lower Extremity Functional Scale (LEFS). Subgroup analyses were evaluated for sex, phenotype, and type of physical activity practiced during the 3-year period. RESULTS: For the whole group, there was a significant decline in the scores of the Mini-BESTest and the LEFS. Also, MIMS significantly declined for prehension, lateral pinch as well as for hip abductors, knee extensors and ankle dorsiflexors muscle groups. Subgroups analyses revealed that men lost more MIMS than women, and that adult phenotype lost more MIMS than juvenile phenotype. CONCLUSION: Quantified muscle testing is a better indicator of disease progression over a 3-year period than functional tests. Phenotype and sex are important factors that influence the progression of DM1.

Identification of a novel TLR5 agonist derived from the P97 protein of Mycoplasma hyopneumoniae.

By modulating specific immune responses against antigens, adjuvants are used in many vaccine preparations to enhance protective immunity. The C-terminal domain of the protein P97 (P97c) of Mycoplasma hyopneumoniae, which is the etiologic agent of porcine enzootic pneumonia, has been shown to increase the specific humoral response against an antigen when this antigen is merged with P97c and delivered by adenovectors. However, the immunostimulating mechanism of this protein remains unknown. In the present study, recombinantly expressed P97c triggered a concentration-dependent TLR5 activation and stimulates the production of interleukin-8 from HEK-Blue mTLR5 cells. Circular dichroism spectroscopy and prediction of 3-dimensional conformation exposed a relevant secondary and tertiary structural homology between P97c and flagellin, the known potent TLR5 agonist. P97c adjuvanticity was evaluated by fusing the conserved epitope of the ectodomain matrix 2 protein (M2e) of the influenza A virus to the protein. Mice immunized with P97c-3M2e revealed a high antibody titer against the M2e epitope associated with a mixed Th1/Th2 immune response. Overall, this study identifies a novel agonist of the pattern recognition receptor TLR5 and reveals that P97c is a potential adjuvant through the activation of the innate immune system.

Nanoparticle-Based Vaccines Against Respiratory Viruses.

The respiratory mucosa is the primary portal of entry for numerous viruses such as the respiratory syncytial virus, the influenza virus and the parainfluenza virus. These pathogens initially infect the upper respiratory tract and then reach the lower respiratory tract, leading to diseases. Vaccination is an affordable way to control the pathogenicity of viruses and constitutes the strategy of choice to fight against infections, including those leading to pulmonary diseases. Conventional vaccines based on live-attenuated pathogens present a risk of reversion to pathogenic virulence while inactivated pathogen vaccines often lead to a weak immune response. Subunit vaccines were developed to overcome these issues. However, these vaccines may suffer from a limited immunogenicity and, in most cases, the protection induced is only partial. A new generation of vaccines based on nanoparticles has shown great potential to address most of the limitations of conventional and subunit vaccines. This is due to recent advances in chemical and biological engineering, which allow the design of nanoparticles with a precise control over the size, shape, functionality and surface properties, leading to enhanced antigen presentation and strong immunogenicity. This short review provides an overview of the advantages associated with the use of nanoparticles as vaccine delivery platforms to immunize against respiratory viruses and highlights relevant examples demonstrating their potential as safe, effective and affordable vaccines.

Engineering and evaluation of amyloid assemblies as a nanovaccine against the Chikungunya virus.

The design of nanoparticles exposing a high density of antigens constitutes a promising strategy to address safety concerns of conventional life-attenuated vaccines as well as to increase the immunogenicity of subunit vaccines. In this study, we developed a fully synthetic nanovaccine based on an amyloid peptide sequence with high self-assembling properties. The immunogenic epitope E2EP3 from the E2 glycoprotein of the Chikungunya virus was used to evaluate the potential of a 10-mer peptide derived from an endogenous amyloidogenic polypeptide as a novel vaccine platform. Chimeric peptides, comprising the peptide antigen attached to the amyloid core by a short flexible linker, were prepared by solid phase synthesis. As observed using atomic force microscopy, these polypeptides self-assembled into linear and unbranched fibrils with a diameter ranging from 6 to 8 nm. A quaternary conformation rich in cross-ß-sheets characterized these assemblies, as demonstrated by circular dichroism spectroscopy and thioflavin T fluorescence. ELISA assays and transmission electronic microscopy of immunogold labeled-fibrils revealed a high density of the Chikungunya virus E2 glycoprotein derived epitope exposed on the fibril surface. These amyloid fibrils were cytocompatible and were efficiently uptaken by macrophages. Mice immunization revealed a robust IgG response against the E2EP3 epitope, which was dependent on self-assembly and did not require co-injection of the Alhydrogel adjuvant. These results indicate that cross-ß-sheet amyloid assemblies constitute suitable synthetic self-adjuvanted assemblies to anchor antigenic determinants and to increase the immunogenicity of peptide epitopes.